From a broad perspective, the proposed studies will investigate how conceptually distinct molecular events, such as transcription and splicing, can be integrated into one system. This concept forms the foundation behind the proposed investigations into the mechanisms underlying RNA polymerase II (RNAP II) regulation of pre-mRNA splicing. RNAP II is a central component of the gene regulatory system because it is directly responsible for the synthesis of mRNA from genomic templates. Consequently, the regulation of RNAP II activity is critical to many aspects of human health and disease including developmental biology, cancer, and gene therapy. Surprisingly, the extent to which RNAP II is involved in the expression of genes appears to extend well beyond transcription. Highly purified preparations of RNAP II and other splicing factors will be utilized in splicing assays in vitro to characterize the molecular interactions between RNAP II and the splicing machinery.